Human disorders caused by neuronal ion channel dysfunction are a major source of pain, suffering, and economic hardship. Their amelioration can be greatly facilitated by understanding the normal in vivo physiological functions served by the large number of biophysically distinct ion channels present in all animals. Our long-term goal is the development of a generally-applicable transgenic toolkit that will permit the testing of hypotheses concerning the physiological functions of particular ion channel subtypes in specific neural circuits in intact behaving animals. Our approach is based on the "tethered toxin" technology, wherein peptide ion channel blockers from venomous predators are expressed as fusion proteins tethered to the extracellular side of the plasma membrane. Preliminary studies applying this approach indicate that tethered spider toxins function as cell-autonomous ion channel blockers with their expected target selectivity when expressed in specific neuronal circuits in the brains of transgenic Drosophila melanogaster fruit flies. Preliminary studies also indicate that the venoms of Australian funnel-web spiders of the Atracinae family contain a vast diversity of uncharacterized peptide toxins expected to target a wide variety of ion channel subtypes. The proposed aims are thus directed at (1) identifying novel spider toxins with high potency against neuronal ion channels and (2) determining the molecular identities of the ion channel targets of each identified toxin. The first aim will be achieved by screening for specific behavioral effects of expressing numerous different tethered funnel-web spider toxins in behavioral control circuits in the brains of transgenic flies. The second aim will be achieved by using a combination of in vitro and in vivo electrophysiological approaches to identify the molecular target(s) of each toxin identified in the first aim. The proposed research will enable the entire Drosophila neurobiology community to begin to test hypotheses concerning the roles of particular ion channel subtypes in specific neural circuits in intact behaving animals that have been refractory to traditional approaches. Because of the extensive conservation of biophysical and physiological mechanisms of neuronal function between flies and mammals, the novel toxins we identify will not only be of tremendous use for in vivo fly neurobiology, but also as pharmacological reagents for probing the structure and function of mammalian ion channels in health and disease. [unreadable] [unreadable]